Insights into the genome of Methylobacterium sp. NMS14P, a novel bacterium for growth promotion of maize, chili, and sugarcane

A novel methylotrophic bacterium designated as NMS14P was isolated from the root of an organic coffee plant (Coffea arabica) in Thailand. The 16S rRNA sequence analysis revealed that this new isolate belongs to the genus Methylobacterium, and its novelty was clarified by genomic and comparative genomic analyses, in which NMS14P exhibited low levels of relatedness with other Methylobacterium-type strains. NMS14P genome consists of a 6,268,579 bp chromosome, accompanied by a 542,519 bp megaplasmid and a 66,590 bp plasmid, namely pNMS14P1 and pNMS14P2, respectively. Several genes conferring plant growth promotion are aggregated on both chromosome and plasmids, including phosphate solubilization, indole-3-acetic acid (IAA) biosynthesis, cytokinins (CKs) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, sulfur-oxidizing activity, trehalose synthesis, and urea metabolism. Furthermore, pangenome analysis showed that NMS14P possessed the highest number of strain-specific genes accounting for 1408 genes, particularly those that are essential for colonization and survival in a wide array of host environments, such as ABC transporter, chemotaxis, quorum sensing, biofilm formation, and biosynthesis of secondary metabolites. In vivo tests have supported that NMS14P significantly promoted the growth and development of maize, chili, and sugarcane. Collectively, NMS14P is proposed as a novel plant growth-promoting Methylobacterium that could potentially be applied to a broad range of host plants as Methylobacterium-based biofertilizers to reduce and ultimately substitute the use of synthetic agrochemicals for sustainable agriculture.


Introduction
The use of agrochemicals has enormous adverse effects on the environment, human and animal health, and soil biodiversity, threatening food security and safety globally. The continuous accumulation of agrochemical residues in soil reverses soil microbial diversity, resulting in the loss of soil fertility, which cannot sustain plant growth and development [1,2]. To diminish agrochemical usage, plant growth-promoting bacteria (PGPB) are proposed as an alternative agent or "biofertilizer" to promote the resilience of the agroecosystem. The term "biofertilizer" has been applied for the use of living microorganisms which, when applied to seeds, plant surfaces, or soils, can live together with plants and stimulate the growth of the host by increasing the bioavailability of primary nutrients and providing beneficial traits [3]. Important characteristics of several PGPB include their capability to perform biological nitrogen fixation, phosphate solubilization, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and production of siderophores and phytohormones [4]. The interest in applying PGPB as biofertilizers has been increasing as a sustainable substitute for the use of agrochemicals in crop production. However, ecological and geographical factors have been known to have a significant impact on the sustainable establishment of PGPB [5]. Therefore, the discovery of the local strains could be a key success factor for substantial use as biofertilizers that are eco-friendly and improve soil fertility for each specific region.
Several members of the genus Methylobacterium have been reported for their plant growth-promoting (PGP) activities, including M. oryzae, M. nodulans, M. radiotolerans, and some unclassified species of the Methylobacterium genus [6][7][8], implying their importance as microbial-based biofertilizers. They can utilize organic one-carbon (C1) compounds as the sole source of carbon and energy, especially methanol. In association with plants, Methylobacterium gains benefit from methanol which is emitted by the host plant as a by-product of the cell wall synthesis [9]. Methylobacterium is normally found in the phyllosphere, however, some have been isolated from roots, shoots, seeds, and seedlings [10]. In nature, Methylobacterium usually co-occurs with other bacteria such as Pseudomonas spp. and Sphigomonas spp. [11] and arbuscular mycorrhizal fungi (AMF) [12]. This indicates that Methylobacterium commonly exists as a member of a consortium that can adapt to a variety of environmental conditions and perform several tasks of plant growth stimulation, i.e., plant nutrient support, plant hormone production, and pathogen inhibition [13,14].
In this study, Methylobacterium spp. were isolated from organic coffee roots by imprinting methods. One of the isolates designated as Methylobacterium sp. NMS14P (hereafter referred to as NMS14P) revealed significant PGP activities as observed from the various plant phenotypes (maize, chili, and sugarcane), which included root development, height, and total biomass. Maize, chili, and sugarcane are economical crops of Thailand, which require a lot of chemical fertilizers and pesticides to increase their productivity. Therefore, the success in the application of NMS14P as a potential native PGPB could provide an alternative way for sustainable agriculture management. To evaluate the functional capabilities of NMS14P as a PGPB, the whole genome of NMS14P was sequenced and comparatively analyzed with 11 closely related Methylobacterium genomes. The overall genomic information of NMS14P supports and provides insight into the molecular mechanisms underlying the plant growth-promoting characteristics of NMS14P, which could be applied to the development of either a single or consortium of Methylobacterium-based biofertilizers in the future.

Isolation of NMS14P
NMS14P was isolated from the root of coffee (Coffea arabica variety Chiang Mai 80) during the reproductive phase, employing the root-imprinting method as described by Corpe (1985) [15] with some modifications as follows. The coffee roots were collected from an organic coffee plantation at Baan Pa Miang, Lampang, Thailand. The soil particles were removed from the roots manually, and the roots were then washed with sterile distilled water 5 times. Subsequently, they were air-dried before being placed on nitrate mineral salts agar medium (NMS; ATCC medium 1306) supplemented with 1% (v/v) methanol and cycloheximide (100 μg/ml) to inhibit the growth of fungi. After incubating the plates at 30˚C for 7 days, the pink-pigmented bacterial colonies were picked and purified by re-streaking on NMS agar supplemented with 1% (v/v) methanol until a single isolate was obtained. The selected colony designated as NMS14P was then kept in 20% glycerol for further characterization.

Preparation of NMS14P inoculum
NMS14P was grown on NMS agar supplemented with 1% methanol at 30˚C. After 7 days of incubation, the NMS14P lawn was scraped and resuspended in sterile distilled water. The cell suspension was inoculated into a 1/4 strength of nutrient broth (Difco, USA) at OD600 of 0.2 and cultured to the mid-log phase at 25˚C with shaking at 150 rpm. The cell pellet was collected by centrifugation at 8,000g for 15 min, washed twice, and resuspended in sterile distilled water. NMS14P solution at OD600 of 0.3 (approximately 10 8 CFU/ml) was used as an inoculum at a final concentration of 10 6 CFU per gram of sterile soil.
Effects of NMS14P inoculation on the growth of maize (Zea mays L.), chili (Capsicum annuum), and sugarcane (Saccharum officinarum L.) The effect of NMS14P inoculation on plant growth was observed by using maize, chili, and sugarcane as plant models. The pot experiments were conducted in a controlled growth room (26±4˚C temperature, 72±5% relative humidity, 176.56±47.09 μmol.m −2 .s −1 brightness, and 16/8-h light/dark photoperiod), located at the King Mongkut's University of Technology Thonburi, Bangkhuntien Campus. All plants were grown in a soil mixture consisting of peat moss and perlite at a 1:1 (v/v) ratio and then inoculated with NMS14P suspension, defined as a treatment, while the controls were treated with sterile distilled water. For maize, the seeds (genotype PAC339; Pacific Seeds, Thailand) were surface sterilized by rinsing with 150 ml of sterile distilled water (1 min, 3 times), and soaking for 5 min in 100 ml of 2% sodium hypochlorite (NaClO) solution (Sigma Aldrich, USA), then rinsing with 150 ml of sterile distilled water, followed by immersing in 70% (v/v) ethanol (Merck Company Ltd., Germany) for 5 min. The final wash was performed by rinsing with 150 ml of sterile distilled water for 5 min, 5 times. The seeds were air-dried in a biosafety cabinet and subsequently germinated in 6 x 5-inch pots (height x diameter) for approximately 7 days before the treatment. For chili, the chili seeds (variety Superhot 2 F1; East-West Seed, Thailand) were surface sterilized by immersing in 70% (v/v) ethanol for 1 min and followed by immersing in 2% NaClO solution (Sigma Aldrich, USA) for 20 min. Seeds were finally washed in sterile distilled water 5 times before air-dried in a biosafety cabinet. The sterilized seeds were sown in a 30 g sterile peat moss: perlite mixture (1:1; v/v) in a 50 ml falcon tube. After 4 weeks of germination, seedlings were transferred to 4 x 6-inch pots (height x diameter) filled with 350 g of sterile peat moss: perlite mixture (1:1; v/v). For sugarcane, the plantlets (variety Khon Kaen 3 (KK3); obtained from the plant propagation center No.10 Udon Thani Province, Thailand) were transferred to peat moss: perlite mixture (1:1; v/v) in 6 x 5-inch pots (height x diameter) and grown for approximately 4 weeks before the treatment. The in vivo experiments were carried out with 3, 4, and 5 replicates for sugarcane, chili, and maize, respectively.
The plants were regularly watered every two days with 100 ml of sterile distilled water. NMS14P suspension at a final concentration of 10 6 CFU/g soil was applied as a soil inoculum during the first week of plantation and every two weeks until the end of the experiments. The sterile distilled water was used in the control plants. Plant growth was determined by measuring cumulative plant height, number of leaves, and shoot and root dry weight at 35, 75, and 56 days after inoculation for maize, chili, and sugarcane, respectively. Shoot and root dry weights were obtained after oven drying at 70˚C until a constant weight was achieved.
Statistical analysis of plant growth-promoting experiment was carried out by t-test using R program v.3.6.3 [16] and subsequently visualized with ggplot2 R package [17].

Inorganic phosphate solubilization test on Pikovskaya's (PVK) agar
NMS14P was streaked on Pikovskaya's (PVK) agar plate containing tricalcium phosphate as an inorganic phosphate source and incubated at 30˚C for 7 days. Inorganic phosphate solubilization activity was observed from the clear zone around the colony [18].

Alkaline phosphatase activity assay
The alkaline phosphatase activity of NMS14P was determined using the Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System for ELISA (Sigma-Aldrich) following the instruction from the manufacturer with some modifications as follows. A total of 200 ml of NMS14P suspension was centrifuged and the supernatant was discarded. The cell pellet was resuspended in 400 μl of pNPP substrate and incubated for 24 hours in a dark condition at room temperature. The reaction was stopped with 100 μl of 3M NaOH solution. The reaction mix was centrifuged to discard the bacterial cells, and supernatant color was observed. The presence of alkaline phosphatase activity was determined from the yellow color of the reaction compared to the colorless of the control, which did not contain bacterial cells [19].

Urease activity assay
NMS14P was grown on NMS agar supplemented with 1% methanol at 30˚C for 7 days. The lawn of the colony was scraped and suspended in sterile distilled water. Urease activity was determined by the colorimetric method following Tanaka et al. (2003) [20] with some modifications. A urea buffer solution containing 3% urea in phosphate buffer with pH 7 and 0.001% phenol red as an indicator was prepared. NMS14P cell was then suspended in this urease buffer solution and incubated at room temperature for 24 hours. The color change was observed by comparing it with the control reaction which had no bacterial cells. The color change from light orange to magenta in the urea buffer solution indicated positive urease activity.

1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity test
ACC deaminase activity was determined following Penrose and Glick (2008) [21] with some modifications. NMS14P was cultured in a Dworkin-Foster (DF) salt minimal medium supplemented with 5 mM (NH 4 ) 2 SO 4 as a nitrogen source for non-ACC-induced control and 3.0 mM ACC as a nitrogen source for ACC-induced cells. The ACC solution was sterilized through a 0.2 μM membrane filter. Cell suspension of NMS14P was inoculated into the medium and incubated at 25˚C with shaking at 150 rpm for 7 days. A 5 ml cell was harvested by centrifugation at 8,000 g for 10 min. The cells were then washed with 0.1 M Tris-HCl, pH 7.6. A 1 ml cell suspension was transferred to a 1.5 ml microcentrifuge tube and centrifuged at 13,000 g for 5 min before resuspending in 600 μl of 0.1 M Tris-HCl, pH 8.5. The cell suspension was extracted by adding 30 μl of toluene and vortexed for 30 s. The toluenized cell suspension was subjected to an ACC deaminase activity test. To detect the by-product, αketobutyrate, of the ACC deaminase reaction, 20 μl of 0.5 M ACC was added to the 200 μl of toluenized cell suspension, and then incubated at 30˚C for 15 min. After that, 1 ml of 0.56 N HCl was added to the reaction, vortexed, and then centrifuged for 5 min at 13,000 rpm at room temperature. A 1 ml supernatant was mixed with 800 μl of 0.56 N HCl in a glass tube and added with 300 μl of 2,4-dinitrophenylhydrazine reagent (0.2% 2,4-dinitrophenylhydrazine in 2 N HCl), then vortexed before incubated at 30˚C for 30 min. A total of 2 ml of 2 N NaOH was added and mixed. The reaction containing α-ketobutyrate and 2,4-DNP-hydrazone produced a brown solution. The amount of α-ketobutyrate from the reaction was determined by measuring its absorbance at 540 nm and compared with the blank control and non-ACC-induced cell [21].

Indole acetic acid (IAA) biosynthesis determination
To determine the IAA biosynthesis of NMS14P, a single colony of NMS14P was inoculated in 5 ml of half-strength Tryptic Soy broth (TSB) supplemented with 5, 10, and 20 mM L-tryptophan and incubated at 25˚C with shaking at 150 rpm for 6 days. The supernatant was collected by centrifugation at 8,000 g for 10 min. IAA was detected by the colorimetric method as described by Gilbert et al. (2018) [22] with slight modifications. The Salkowski reagent was added to the supernatant at a 2:1 ratio (v/v) (Salkowski reagent: Supernatant) in triplicate. The reactions were incubated in a dark condition at room temperature for 30 min. The concentration of IAA production was measured at the wavelength of 530 nm and then compared with the IAA standard curve.

Hypersensitivity reaction (HR) assay
Hypersensitivity reaction (HR) assay was performed as described by Huang et al. (1988) [23] with some modifications. Briefly, the bacterial cell suspension at the OD600 of 0.3 was prepared in sterile distilled water. Tomato (Solanum lycopersicum L.) leaves were wounded by a 27-gauge needle underneath the leaves and then infused with 10 μl of sterile distilled water, Pseudomonas aeruginosa suspension, and Methylobacterium sp. MNS14P suspension for the negative control, positive control, and NMS14P, respectively. The infiltrated leaves were put on wet cotton in a sterilized 90 mm petri dish and incubated at room temperature (12-h light/ dark cycle). The development of HR was observed every day.

Genomic DNA extraction and sequencing
Genomic DNA (gDNA) of NMS14P was extracted using the ZymoBIOMICS MagBead DNA kit according to the manufacturer's instructions (Zymo Research, USA). The quality and quantity of the extracted gDNA were further examined with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, United States). A total of 2 μg of genomic DNA was sent to Novogen AIT Singapore for paired-end (2 x 150 bp) sequencing using a Nextera XT sequencing kit on Illumina NovaSeq 6000. For nanopore long-read sequencing, 1.2 μg of genomic DNA was subjected to library preparation using an SQK-LSK109 ligation kit (Nanopore, Oxford, UK), and sequencing was performed in-house on the MinION R9 flow cell (Nanopore, Oxford, UK), according to the manufacturer's protocol.

Genomic islands, pathogenic potential, virulence, and antimicrobial resistance (AMR) genes predictions
Genomics islands were computationally predicted using IslandViewer 4 [46]. The pathogenic potential of NMS14P was evaluated using PathogenFinder v.1.1 web service with automated mode [47]. The putative virulence factors were assessed using BLASTp search against protein sequences from the full dataset in the virulence factor database (VFDB) [48]. The presence of AMR genes was identified with a web server of Resistance Gene Identifier (RGI) v.5.1.1 module of Comprehensive Antibiotic Resistance Database (CARD) v.3.1.1 [49].

Bacterial species identification and classification
Bacterial species identification was performed using 16S rRNA gene and overall genome relative index (OGRI) methods. For 16S rRNA-based identification, it was done by directly amplifying the 16S rRNA gene from the NMS14P single isolated colony by colony PCR using universal primers 27F and 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'). PCR product was further purified and sequenced with the Sanger sequencing technique with the 1492R primer. The output sequence was inspected, and low-quality bases were trimmed using Sequence Scanner Software v2.0 (ThermoFisher Scientific) prior to downstream analyses (S1 Text). A high-quality sequence was then used as a query for blast search using NCBI BLASTn (https://blast.ncbi.nlm.nih.gov). Additionally, the Sanger sequencing-derived 16S rRNA gene sequence was also used to ensure the authenticity of the genome assembly by pairwise alignment with the predicted 16S rRNA sequences from the assembled genome [50]. Taxonomic classification based on OGRI was done with two approaches as follows. Firstly, a whole-genome-based taxonomic analysis was carried out using the Type (Strain) Genome Server (TYGS), an open-source bioinformatics platform available under https://tygs.dsmz.de [51]. 16S rRNA-based phylogenetic and phylogenomic trees were inferred with BLAST distance as implemented in TYGS and visualized using the ggtree R package [52]. Secondly, a core genome alignment method as proposed by Chung et al. (2018) [53] was employed to identify a set of orthologous sequences conserved in all aligned genomes. In addition, the genomic similarities between strain NMS14P and other closely related species (Table 1) were determined using the average nucleotide identity (ANI) algorithm with Fas-tANI v.1.32 [54]. Core genome alignment similarity index (CGASI) and ANI matrices were visualized with the ggplot2 R package [17].

Comparative genomic analysis
To avoid comparison biases caused by different annotation tools, all reference genomes downloaded from Refseq NCBI (Table 1) were re-annotated with Prokka v.1.14.5 [35]. Pangenome analysis was subsequently carried out using Roary v.3.13.0 [55] with the minimum percentage identity for BLASTp set to 90. Single representative sequences from each of the clusters in the pangenome were further aligned against the KEGG database using GhostKOALA [40], while COG and custom Methylobacterium database annotations were executed with DIAMOND BLASTx v.0.9.24 [41]. The analysis results were then visualized using the R program v.3.6.3 [16]. The comparison of 12 Methylobacterium genomes was executed with CGView Comparison Tools (CCT) v.1.0.2 [56].
The overall method performed in this study was summarized in a flowchart and is provided in the S1 Fig.

In vivo plant growth-promoting activity test of NMS14P
NMS14P was isolated from the organic coffee roots by the root imprinting method. To assess the PGP traits of NMS14P, in vivo tests were carried out with three economically important plants, namely maize, chili, and sugarcane under a greenhouse environment (S2 Fig). As shown in Fig 1A, the shoot dry weight and shoot height of NMS14P-inoculated maize were significantly greater than the control plants (p < 0.05). In chili, inoculation of NMS14P revealed significantly higher shoot dry weight and the number of leaves than the control plants (p < 0.05) (Fig 1B). NMS14P also significantly stimulated the growth of sugarcane in terms of cumulative plant height when compared with control plants (p < 0.05) (Fig 1C). These results indicated that a single strain treatment of NMS14P could substantially promote the growth of both monocot and dicot plants of commercial importance. It was thus hypothesized that NMS14P might be able to provide extra available nutrients by mobilizing the essential building blocks from the soil and environments and function as a biofertilizer for plant growth and development. In addition, applications of NMS14P in maize, chili, and sugarcane under a greenhouse environment revealed the possible broad host range of PGP activities of this bacterial species. Besides PGP activities, the hypersensitivity reaction (HR) assay revealed that no necrotic response was found on the NMS14P-infiltrated leaves of the tomato plant model as shown in the S3 Fig. Altogether, it is interesting to investigate the molecular mechanisms underlying these PGP traits at the genomic level. Thus, the genome of NMS14P was further characterized to identify the functional capabilities of the genes governing its PGP attributes.

General information on the genomic dataset
A total of 572,350 and 10,270,449 raw reads were generated by the Nanopore and Illumina sequencing platforms, respectively (S1 Table). After removing low-quality bases, 0.97% of Nanopore reads were filtered out, while 0.94% of reads were discarded for Illumina. The remaining sequences were 555,629 with an average Phred score of 9.3 and mean read length of 5,394.2 and 9,671,658 paired-end reads with an average Phred score of 36 and mean read length of 149 for Nanopore and Illumina, respectively (S1 Table). These high-quality sequences were used for further genome assembly steps.

Genome assembly and quality assessment of NMS14P
The whole-genome sequences of NMS14P were obtained by the combination of Illumina paired-end and Nanopore sequencing platforms. Genome assembly was performed with Trycycler [26] by combining several assembler programs, including Flye [27], Miniasm [28], Minipolish [29], and Raven [30] to get a long-read assembly consensus, which was further polished with high-quality Illumina short-reads using Pilon [31]. Genome completeness and accuracy were estimated using BUSCO [34], CheckM [32], and QUAST [33]. The quality of the assembled genome revealed completeness of more than 99.5%, contamination of 0.63%, and 743X average depth coverage (S2 Table), implying a high-quality assembled genome. Moreover, a high identity of 16S rRNA sequences derived from Sanger sequencing compared with the ones extracted from the whole genome assembly (S1 Text) confirmed the authenticity of genomic data [50].

General characteristics of the NMS14P genome
A high-quality complete genome was generated for NMS14P, comprising a single circular chromosome of 6,268,579 bp with a high G+C content of 71.12%, and two plasmids labeled as pNMS14P1 (542,519 bp; considered as a megaplasmid) and pNMS14P2 (66,590 bp) (Fig 2), possessing overall G+C contents of 70.61% and 64.62%, respectively (Table 2). A total of 5,879 protein-coding sequences (CDSs) with an average length of 914 bp, 12 rRNAs, and 73 tRNAs were predicted from the NMS14P chromosome, of which 918 (15.61%) of CDSs were assigned as proteins with unknown or hypothetical function (Table 2). Furthermore, 496 CDSs with an average length of 904 bp were identified in pNMS14P1, and 113 CDSs with an average length of 463 bp were found in pNMS14P2 (

Identification of NMS14P as a novel species
NMS14P was taxonomically identified using both full-length 16S rRNA gene and complete genome information, including digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and core genome alignment similarity index (CGASI). The position of NMS14P in both 16S rRNA-based phylogenetic and phylogenomic trees was consistent and revealed that this species was evolutionarily separated from the closely related M. organophilum DSM 760 and M. radiotolerans JCM 2831 (Fig 3). Furthermore, NMS14P showed low similarity scores with other Methylobacterium-type strains, which were less than the species cutoff of 70% and 95% for dDDH (formula d4) ( Table 3) and ANI (Fig 4), respectively. NMS14P also exhibited CGASI values lower than the 96.8% identity threshold of bacterial species boundary (Fig 4) against 11 reference genomes in the same genus, and a paraphyletic clade was not observed (S4 Fig). Therefore, these results indicated that NMS14P is a new Methylobacterium species.

Genomic islands and preliminary safety assessment of NMS14P
A total of 118 genomic islands (GIs) were identified by IslandViewer 4 [46] throughout the NMS14P genome (S4 Table). Particularly, 115 and 3 GIs were discovered in the chromosome (1,253,005 bp, 19.99% of the entire chromosome) and pNMS14P1 (19,182 bp, 3.54% of the overall pNMS14P1), respectively. The size of these putative islands ranged from 4,025 bp to 54,755 bp. The largest GI (GI-013) possessed 50 genes, whereas the smallest GI (GI-048) contained 12 genes. The majority of the predicted GIs were genes encoding for hypothetical proteins (811 genes), followed by tyrosine recombinase XerC (18 genes), sensor histidine kinase RcsC (12 genes), IS5 family transposase ISMex40 (10 genes), IS630 family transposase ISMex30 (9 genes), D-inositol-3-phosphate glycosyltransferase (7 genes), IS3 family transposase ISMtsp5 (7 genes), protein-methionine-sulfoxide reductase catalytic subunit MsrP (5 genes), and other products with numbers of genes less than 5 (S4 Table). Notably, three GIs, i.e., GI-059, GI-070, and GI-079, were recognized as genes to potentially confer the adaptability and competitive traits of this isolate in the rhizosphere environment. The pathogenic potential of NMS14P was assessed using PathogenFinder [47]. This webbased tool predicted NMS14 as a non-human pathogen with a low probability score of 0.19 (S5 Table). The presence of virulence factors in the NMS14P genome was identified using the virulence factors of the pathogenic bacteria database (VFDB) [48]. Accordingly, 11 and 2 hits were found for genes designated as virulence factors located in the chromosome and

PLOS ONE
Insights into the genome of Methylobacterium sp. NMS14P pNMS14P1, respectively (S6 Table). In addition, antimicrobial resistance (AMR) genes were screened using the CARD pipeline using the strict mode [49]. Evaluation of resistomes in the genome exhibited that NMS1P possessed four copies of adeF, an AMR gene in the family of resistance-nodulation-cell division (RND) antibiotic efflux pump (S7 Table).

Comparative genomic analysis of NMS14P with closely related strains
The genome BLAST comparison of NMS14P against all reference genomes was carried out with CGView Comparison Tools (CCT) [56] using 11 closely related strains with complete, scaffold, or contig-level genomes ( Table 1). The BLAST comparison revealed a high level of heterogeneity among the strains, as indicated by light blue colors representing BLAST hit less than 40% similarity (Fig 5). To further analyze the unique regions as well as conserved genes in these genomes, pangenome analysis was performed using the Roary program [55]. The result exhibited a steadily increased number of pangenomes with the sequential addition of each new genome (Fig 6A). In contrast, the size of core genes decreased with each addition of a new genome and finally reached a plateau state with the addition of the fifth genome and stayed nearly constant until the last genome addition (Fig 6A).
A total of 14,291 orthologous groups were discovered, consisting of 3,176 core genes shared by every strain. Several genes involved in methanol oxidation were identified as the core, including the mxa gene family (mxaACDFGIJKL), xoxF, and pqqBCEL. In addition, a set of genes (gckA, mdh, eno, mcl, ppc, mtkAB, hprA, AGXT, and glyA) involved in formaldehyde assimilation via serine pathway (KEGG Module: M00346) was also found to be conserved in all Methylobacterium genomes in this study. Furthermore, 5,920 dispensable genes and 1,408 genes unique to strain NMS14P were observed, exhibiting the largest number of strain-specific genes among others, while JCM 2831 was the lowest with 11 strain-specific genes (Fig 6B). These NMS14P-unique genes were predominated by unannotated sequences (hypothetical proteins) according to Prokka, KEGG, COG, and custom database annotation (67.19%) (S5 Fig) and might represent candidate putative novel genes, while the rests were genes that are participated in metabolic pathways (56 genes), two-component system (22 genes),

Identification of plant growth-promoting genes in NMS14P
Potential capabilities of NMS14P for enhancing nutrient availability to plants. Nitrogen (N) is an essential microelement for plant growth and development. Plants uptake nitrogen in the form of NH4 + (ammonium) or NO3 -(nitrate). NMS14P genome contained a urease gene cluster ureABCDEFGHJ, nitrate reductase (nasAB), and nitrite reductase (nirA),

PLOS ONE
which are involved in the plant-available N support. Nitrate can be converted into ammonia through pathways of assimilatory nitrate reduction (ANRA) and dissimilatory nitrate reduction (DNRA). The ANRA pathway consists of two steps where nitrate is first reduced to nitrite by nitrate reductase (NasAB, NR, or NarB) and is then reduced to ammonia by nitrite reductase (NirA or NIT-6). The presence of nitrate/nitrite transporter (nrt), nitrate reductase (nasAB), and ferredoxin-nitrite reductase (nirA) genes in the NMS14P genome suggested that this strain could also reduce nitrate into ammonia via the ANRA pathway. Phosphorus (P) is an indispensable element involved in a plethora of biological and biochemical mechanisms which determine plant growth and yield [58]. Although a large amount

PLOS ONE
Insights into the genome of Methylobacterium sp. NMS14P of P is present in the soil; however, only a small portion is available for plants due to the formation of insoluble organic and inorganic phosphate complexes [59]. Also, the issue of climate change has influenced the availability, acquisition, and translocation of P, creating new challenges in P management [60]. Phosphate (P) solubilization is a crucial attribute to enhance the bioavailability and uptake of essential nutrients for plant growth and development. NMS14P genome contained genes related to P solubilization, especially inorganic P solubilization, including pyrophosphatase (ppa), exopolyphosphatase (ppx), and glucose dehydrogenase
Another vital element for plant growth and development besides N, P, and K is sulfur (S). It plays a role in photosynthesis, respiration, and the formation of chlorophyll and cell membrane structure in plants, which are related to the quality and yield of crop production [61][62][63]. Plants absorb S as sulfate from the soil, however, soil environments contain sulfate only 1-5% of the total bioavailable sulfur [64]. The sulfur-oxidizing (SOX) system encoded by Sox-ACDXYZ genes was found in the NMS14P genome. The SOX complex is involved in thiosulfate, sulfide, sulfite, and elemental sulfur oxidation and generates sulfate as the final product [65]. The presence of the SOX system supported that NMS14P could increase plant-available S in soil. However, S-sulfosulfanyl-L-cysteine sulfohydrolase (soxB) was not found in the genome. The absence of this gene could be further investigated to complete the function of the SOX complex in this NMS14P.
Potential capabilities of NMS14P in phytohormone production. Seven genes (trpABC-DEFG) involved in the biosynthesis of L-tryptophan, the precursor for auxin biosynthesis, were found in the NMS14P genome. Indole-3-acetaldehyde dehydrogenase, amidase, and nitrilase encoded by ALDH, amiE, and nth A-B genes, respectively, were also identified in the genome, suggesting that NMS14P might be able to produce auxin via indole-3-acetonitrile (IAN) pathway. NMS14P genome also carried the miaA gene, which is responsible for the production of zeatin, one type of cytokinins, through isopentenylation of a specific adenine in tRNAs. These phytohormones are not only crucial for various growth and developmental processes in plants but also play a role in the plant-microbe interaction [66].

PLOS ONE
Insights into the genome of Methylobacterium sp. NMS14P

Potential capabilities of NMS14P to assist plants withstand environmental stresses.
Like many other PGPB, NMS14P contained genes that help plants to tolerate environmental stresses. These were 1-aminocyclopropane-1-carboxylate (ACC) deaminase, trehalose-6phosphate synthase (ostA), and trehalose-6-phosphate phosphatase (ostB). ACC deaminase can degrade ACC, which is the precursor of ethylene biosynthesis [67], while the last two genes were involved in trehalose synthesis.
Potential biocontrol agents of NMS14P for plant disease protection. Bacterial flagellin can be recognized by plant flagellin sensing receptors such as FLS2 and FLS3 depending on plant types, and then triggers plant immune responses and the production of host-defense proteins, which can protect plants from fungal and bacterial infections [68][69][70]. A total of 25 genes involved in the flagellar assembly, including FlgBCDEFGHIK, FlhAB, FliFGIMNPQRY, FlrC, MotAB, and RpoDN were detected in the NMS14P genome. In addition, NMS14P also contained a phenazine biosynthesis gene (PhzF).

Verification of observed plant growth-promoting activities in NMS14P according to the genome information
The potential plant growth-promoting activity of NMS14P was demonstrated by experimental assays. Pikovskaya's (PKV) agar and the alkaline phosphatase yellow (pNPP) liquid substrate system were used to test inorganic and organic phosphate solubilizations, respectively. The clear zone shown only beneath the colony demonstrated that NMS14P could slightly solubilize tricalcium phosphate (Fig 7A). In the case of organic phosphate, NMS14P could produce alkaline phosphatase, which could hydrolyze colorless p-nitrophenyl phosphate (pNPP) to p-nitrophenol (yellow color) (Fig 7B). Urease activity was measured using a colorimetric method with phenol red as an indicator. The presence of urease was indicated by the color change of phenol red from light orange to magenta caused by ammonia production, which increased the pH in the solution (Fig 7C). 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity of NMS14P was induced by ACC addition in a DF salt minimal medium. Toluenized cell extract of isolate grown in DF salt minimal medium with ammonium sulfate (NH 4 ) 2 SO 4 as the sole nitrogen source was unable to degrade ACC as shown by the yellow color ( Fig 7D). However, NMS14P cell extracts grown in a DF medium with ACC as the sole nitrogen source could degrade ACC to ammonia and α-ketobutyrate. After that, α-ketobutyrate derivatized by 2,4-dinitrophenylhydrazine (2,4-DNPH) reagent into α-ketoglutarate 2,4-DNP-hydrazone and then reacted with sodium hydroxide (NaOH) which changed the color of solution into brown (Fig 7D). IAA was detected in the NMS14P culture broth (Fig 7E). NMS14P grown in a half strength of TSB medium with supplementation of 5, 10, and 20 mM of L-tryptophane showed an increase in IAA production following the rise of tryptophan concentration and time of incubation. Specifically, the IAA production was 1.58, 2.46, and 3.30 μg/ml at 48 hours and increased to 5.19, 7.60, and 14.93 μg/ml at 6 days of incubation. These experimental assays proved that NMS14P had plant growth-promoting activities such as urease, phosphate solubilization, ACC deaminase, and IAA production as were identified from the genome information.

Discussion
Plant growth-promoting bacteria (PGPB) promote plant growth through either a direct mechanism by facilitating resource acquisition such as N, P, K, S, and other essential minerals or in an indirect way by lessening the inhibitory effects of numerous pathogens on plant growth and development in the form of biocontrol agents [67]. Considering their capabilities for improving crop health and productivity in an environment-friendly manner, they serve as an alternative to the application of chemical inputs in agriculture [71,72].
In the present study, a bacterium (NMS14P) was isolated from the root of a coffee plant (Coffea arabica) cultivated organically in Thailand. Taxonomic identification based on 16S rRNA sequence exhibited that this isolate belonged to the genus Methylobacterium, and its novelty was further confirmed with genomic and comparative genomic analyses. The results showed that NMS14P had low levels of relatedness with other Methylobacterium-type strains in the current database, clarifying that NMS14P is a new methylotrophic bacterial species.
In vitro tests using maize, chili, and sugarcane plants inoculated with NMS14P demonstrated that all bioinoculant-treated plants grew and developed better than uninoculated ones, indicating that this isolate had the capability to stimulate plant growth and development. This is supported by the gene contents in the NMS14P genome involving phyto-fertilization and phyto-stimulation, including phosphate solubilization, urea metabolism, sulfur-oxidizing activity, indole-3-acetic acid (IAA) biosynthesis, cytokinins (CKs) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and trehalose synthesis. A hypersensitivity reaction (HR) assay was also performed and revealed that this bacterium did not cause any necrosis to the tomato, implying that NMS14P has no potential to infect plants as a phytopathogen. In addition, preliminary safety assessments according to the presence of genes related to virulence factors and antimicrobial resistance (AMR) using various tools and databases indicated that NMS14P is a non-human pathogen; however, before released to the environment, follow-up studies would be required.

PLOS ONE
NMS14P could promote plant growth and development by enhancing the bioavailability of essential nutrients, including N, P, and S. Although nitrogen-fixing genes (nifHDK) were not found in the genome, NMS14P contained a urease gene cluster, in which its activity has been confirmed by in vitro test using the colorimetric method. Urease activity plays important roles in protein and nucleotide turnover and urea hydrolysis to improve N availability for soil microbes and plants [73,74]. It has been reported that the application of PGPB increased soil urease activity, which converts urea fertilizers into ammonia [75], thus promoting plant growth [76]. Also, nitrate reductase and nitrite reductase genes that are involved in the reduction of nitrate into ammonia through the ANRA pathway were found in the NMS14P genome. Increasing rates of the ANRA pathway would have multiple advantages, including reserved N in the soil, reduced N 2 O emissions, increased farm productivity, reduced water contamination, and reduced use of N fertilizers [77]. Furthermore, the genomic data showed that NMS14P carried genes for P solubilization, thereby contributing to plant phosphate acquisition. The solubilization assay demonstrated that this isolate could solubilize both organic and inorganic phosphate. In addition, the NMS14P genome harbored several genes that participated in the oxidation of thiosulfate, sulfide, sulfite, and elemental sulfur to sulfate as the final product, playing a key role in the cycling of organic sulfate in soils and enhancing plant growth in soils with low S availability [78].
Some PGPB have been known to directly stimulate plant growth through the production of phytohormones, such as indole-3-acetic acid (IAA) [67]. IAA, the main auxin in plants, is a phytohormone that plays an important role in modulating plant growth and development [79]. NMS14P contained genes involved in L-tryptophan biosynthesis and those that encode for indole-3-acetaldehyde dehydrogenase, amidase, and nitrilase, implying that NMS14P might synthesize auxin through indole-3-acetonitrile (IAN) pathway. This result was supported by the previous study that proposed indole-3-acetamide (IAM) and IAN pathways for IAA biosynthesis in Methylobacterium sp. 2A [8]. Auxin production from this isolate has been confirmed by in vitro test using the Salkowski reagent. Furthermore, the miaA gene was also found in the NMS14P genome. It has been reported that miaA is required for zeatin production in Methylobacterium [80]. Auxin and cytokinin can cause plant cell wall loosening and nutrient leakage from host plants, as well as stimulation of plant growth [81].
Plants are subjected to a broad range of biotic and abiotic stresses that can limit crop growth and productivity. For instance, under stress conditions, the ethylene level in a plant is elevated, leading to the reduction of root and shoot growth [82]. ACC deaminase-producing bacteria can help to decrease ethylene levels, thereby increasing plant growth [83]. NMS14P contained genes encoding for ACC deaminase, in which its activity has been confirmed by the in vitro test using a DF salt minimal medium. In addition, NMS14P also harbored genes involved in trehalose biosynthesis. It has been proposed that trehalose produced by beneficial bacteria could trigger the plant-defense system to prevent damage caused by drought [84]. Apart from abiotic stresses, NMS14P possessed genes for synthesizing a globular protein that is a chief constituent of bacterial flagella, called flagellin, which can be recognized by host plants to elicit their immune responses [69]. Likewise, a phenazine biosynthesis gene was also detected in the NMS14P genome. Phenazines are redox-active nitrogen-containing heterocyclic molecules that act as antibiotics and suppress plant pathogenic microbes such as Rhizoctonia [85] and Fusarium [86]. Altogether, these findings suggested the potential benefit of NMS14P in mitigating the deleterious effects of biotic and abiotic stresses on plant growth and development.
The comparative genomic analysis identified a total of 3,176 core genes, which showed a higher number of core genes in comparison with the previous analysis of nine Methylobacterium genomes (2,010 core genes) [87]. As expected, the mxa gene family, xoxF, and pqqBCEL were found in the core genome. mxa gene cluster is essential for methanol oxidation using a pyrroloquinoline quinone (pqq)-dependent methanol dehydrogenase (MDH) enzyme, while xoxF encodes for the alternative MDH, catalyzing the oxidation of methanol to formaldehyde [88]. The formaldehyde is then either assimilated into cell biomass through the serine cycle or oxidized to CO 2 for energy generation in the cofactor tetrahydromethanopterin-dependent pathway [89,90]. On the other hand, mxaF and xoxF have been used as biomarkers in environmental studies in that both are highly conserved among methylotrophs [91,92]. Interestingly, there were two copies of the act gene in the NMS14P genome, while only one copy of this gene was found in the other Methylobaterium genomes in this study. The act gene encodes for the methanol dehydrogenase activator which is involved in the activation of NAD-dependent methanol dehydrogenase (MDH). ACT is an endogenous activator protein, which dramatically enhances the methanol oxidation in Bacillus methanolicus, however, the detailed mechanism remains unclear [93]. Two copies of act genes in the NMS14P genome might have advantageous attributes in the methanol oxidation activity of this strain, which could be further characterized in the future.
To provide beneficial impacts on the host plants, at first, microbial-based inoculants need to competently colonize the external and/or internal parts of plant tissues, and then establish appropriate interactions with the host [94]. Also, competitive attributes to persist and thrive in a new environment against indigenous microorganisms are highly required [95]. On the other hand, plants secrete sugar and amino acid-enriched metabolites into the soil to attract their microbial counterparts [96]. The composition of root exudates varies across plant species, and specific exudates can act as chemical signals by inhibiting or stimulating the growth of adjacent microbes in the vicinity [97,98]. Therefore, only microbes that can gain benefits from the utilization of these compounds can colonize and develop mutual interactions with the host plant. Interestingly, NMS14P harbored several genes needed to exploit plant exudates, including those that are involved in multiple sugar transporter, ribose transporter, and numerous amino acid transporters. Moreover, NMS14P also contained other genes necessitated to successfully invade the host plant and outcompete the existing microbial communities, such as ABC transporters, chemotaxis, quorum sensing, biofilm formation, and biosynthesis of secondary metabolites. It has been reported that these pathways play essential roles in the rhizosphere and rhizoplane colonization [94,99]. Notably, a putative novel gene annotated to encompass the pesticin C-terminal-like domain of uncharacterized proteins (pesticin lyz-like) was found in the chromosome (NMSCH_04528). This subfamily is made up of uncharacterized proteins that have a lysozyme-like domain which is similar to the C-terminal domain of pesticin, an anti-bacterial toxin produced by Yersinia pestis used to kill related bacteria of the same niche [100]. Altogether, the presence of numerous transporters related to root exudates, together with host colonization and rhizocompetence traits, might contribute to the broad host range characteristic of NMS14P.
Colonization in some environments might necessitate an increased general genetic flexibility as a prerequisite for successful adaptation and increased fitness to continuously changing growth conditions. Genomic islands (GIs) might offer a selective advantage by enhancing the adaptability and competitiveness of a bacterial species within a habitat under certain growth conditions through large numbers of gene transfer mechanisms [101]. Compared with the previously reported genome, NMS14P contained much more GIs (113 GIs) than Methylobacterium oryzae CBMB20, in which 25 GIs were identified in its chromosome [87]. A high number of GIs might be related to the capability of this isolate to colonize and survive in various host plants which possess different environmental conditions. Accordingly, the GI-070 contained genes encoding for linear gramicidin synthase (lgrBD), dimodular non-ribosomal peptide synthase (dhbF), ferrichrome outer membrane transporter/phage receptor (fhuA), and a hypothetical protein. This GI was proposed to be involved in the production of gramicidin antibiotics. It has been known that linear gramicidin is a pentadecapeptide antibiotic produced via the non-ribosomal pathway [102]. Furthermore, two copies of Na + /H + antiporter NhaP2 were found in the GI-079. NhaP2 has been reported to play an important role in protecting cells of Vibrio cholerae and Pseudomonas aeruginosa at low pH [103,104]. In addition, genes encoding for iron complex transport systems (i.e., ATP-binding protein, permease protein, and substrate-binding protein), ferri-bacillibactin esterase BesA, TonB-dependent siderophore receptor, and regulatory protein PchR were found in the GI-059, implying that this GI was linked to iron acquisition. Iron is an essential element involved in the oxido-reduction mechanisms in the cell. As a result, GIs encoding siderophore systems could be thought of as fitness islands that increase the adaptability of this isolate in the rhizosphere environment [101]. Taken together, these three GIs might also importantly contribute to the colonization ability of NMS14P to various host plants in this study.

Conclusions
Methylobacterium spp. have been known for their functions as a plant growth stimulator in various environmental conditions. The complete genome of a novel species, Methylobacterium sp. NMS14P, locally isolated from coffee roots in Thailand, revealed numerous characteristics of PGPB, including urease, phosphate solubilization, phytohormones production, ACC deaminase, and potential biological control agents. Several genes involved in colonization and rhizocompetence have also been reported in this genome, implicating its applicability to a wide array of plant hosts. Based on the in vivo test, NMS14P inoculation could promote the growth of both monocot and dicot plants. The results confirmed the potential use of NMS14P as a single-strain Methylobacterium-based biofertilizer. However, the application of a synthetic microbial community with known bacterial strains could be more attractive for better PGP activities. As such, the interaction between NMS14P and other bacterial strains as well as target plants could be further investigated for the development of suitable communities, thereby reducing chemical fertilizers for viable sustainable agriculture.   Table. Putative novel genes found in the NMS14P genome annotated using a conserved domain database (CDD). (XLSX) S1 Text. Pairwise alignment of Sanger-derived 16S rRNA sequence with 16S rRNA sequences extracted from the genome assembly. (DOCX) S1 Graphical abstract. (TIF)